Serum albumin is the most abundant protein in serum, typically present at 45-50 mg/ml. Albumin functions as a “molecular sponge” binding proteins, lipids, and small molecules in the intracellular space (1-3) and has been found to form associations with peptide hormones, serum amyloid A, interferons, glucagons, bradykinin, insulin, and Streptococcal Protein G (4-7) but an extensive list of binding partners, and whether these partners change with disease, has not been investigated. Previous studies have shown a higher recovery of low molecular weight species when removing high molecular weight species under denaturing conditions, further confirming that larger proteins, such as albumin, are binding peptides (8). Furthermore, albumin has been reported to bind to a small number of specific proteins such as paraoxonase 1 (9), alpha-1-acid glycoprotein (10), and clusterin (11) (indirect interaction through paraoxonase 1) and apolipoprotein E12 in serum. Although albumin binding peptides (below 30 kDa) in serum have been studied, the extent of their binding is currently unknown (13). To date, a comprehensive study of the whole proteins bound to albumin has not been carried out. Additionally, there is no documentation of any changes in the protein/peptide composition, ratio or PTM status of the proteins/peptides bound to albumin.
Albumin has been found to change with disease which alters its binding to metals and currently functions as a biomarker for ischemia. A modification of albumin that has previously been identified as a biomarker for myocardial ischemia is the N-terminus N-acetylation of albumin, which decreases the binding affinity of albumin to cobalt and nickel (21-23). Current patents (24,25) cover the usage of this N-terminal modification of albumin for ischemia and have led to a clinical assay for albumin cobalt binding (ACB assay). In addition to the N-terminal modification, the oxidation of albumin has been proposed to be a marker for oxidative stress (26). MALDI-TOF analysis (Matrix Assisted Laser Desorption/Ionization Time-of-Flight) of the albumin in patients with renal impairment and end-stage renal disease show an increase in the MW of albumin with disease (27). Finally, the fatty acid transport function of albumin is modified in atherosclerosis and diabetes (28). In patients with diabetes, the binding capacity of albumin for fatty acids is increased, and in patients with atherosclerosis the capacity is decreased. In conclusion, the evidence the albumin is changing with disease is clear. What has not been investigated or described previously is altered binding of proteins and/or peptides to albumin in serum. The current work is unique because it includes the analysis of intact proteins, degraded proteins, and peptides, without eliminating any mass range. Furthermore, the current work focuses on the changes in the proteins and peptides that bind to albumin, a feature not addressed in any previous literature.